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  Directed evolution of nucleotide-based libraries using lambda exonuclease

Lim, B. N., Choong, Y. S., Ismail, A., Glökler, J., Konthur, Z., & Lim, T. S. (2012). Directed evolution of nucleotide-based libraries using lambda exonuclease. Biotechniques, 53(6), 357-364. doi:10.2144/000113964.

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© 2012 BioTechniques
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 Urheber:
Lim, B. N., Autor
Choong, Y. S., Autor
Ismail, A., Autor
Glökler, J.1, Autor           
Konthur, Z.2, Autor           
Lim, T. S., Autor
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1433550              
2In vitro Ligand Screening (Zoltán Konthur), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1479653              

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 Zusammenfassung: Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2x108 and 6x107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.

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Sprache(n): eng - English
 Datum: 2012-12
 Publikationsstatus: Erschienen
 Seiten: -
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: DOI: 10.2144/000113964
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Titel: Biotechniques
  Andere : Biotechniques
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: [Natick, MA : Eaton Pub. Co.
Seiten: - Band / Heft: 53 (6) Artikelnummer: - Start- / Endseite: 357 - 364 Identifikator: ISSN: 0736-6205
CoNE: https://pure.mpg.de/cone/journals/resource/954925536105